Ongoing outbreaks of avian influenza in humans stress the need for intensified research efforts to fully characterize the pathogenesis during infection. Clinical course in patients and post-mortem pathology have suggested either presence of influenza virus in tissues outside of the respiratory tract, disease secondary to respiratory infection but in tissues that remain virus-free, or both. Animal studies have also demonstrated extra- pulmonary spread of avian influenza viruses, to an extent and with organ-involvement that appear species- dependent. Assessing the exact contribution of non-respiratory organ pathology to cytokine overproduction, a common feature of the disease in humans leading to multi-organ failure, is critical to successful clinical management, as is a better understanding of the factors leading to development of encephalopathy, also commonly seen. Unfortunately, timing of hospitalization after infection, confounding effects of heroic therapeutic efforts, and ethical considerations precluding biopsy of major organs in already severely compromised individuals, necessitate the use of animal models to study early infection in lungs and other major organs likely affected by the infection. Over the past 3 years, we have successfully optimized a macaque model of human influenza infection for genomic applications. For this purpose, we have used a mildly pathogenic human influenza virus (A/Texas/36/91) to carefully characterize the clinical course, pathology, and transcriptional regulation in response to viral mRNA, viral proteins, or to active viral replication. Next, we will infect Cynomolgus macaques with Texas influenza virus containing up to three genes from the 1918 pandemic flu (A/Brevig Mission/1/18), believed to have originated from an avian virus, and with highly pathogenic avian influenza (A/A/Vietnam/1203/04). In addition to the work performed in previous years, we propose to do an in-depth characterization of any pathology in tissues other than the respiratory tract, through the use of systematic microscopic examination, influenza antigen staining, phenotyping of immune cells in situ, and immunohistochemistry staining for markers of inflammatory and immune processes, including the cyclooxygenase cascade, production of nitric oxide by macrophages, induction of hypoxia response elements, angiogenesis, and others. This approach will be supplemented and corroborated by laser capture microdissection of lesions followed by quantitative reverse transcription PCR for corresponding mRNAs. We believe that the combination of several years of direct experience with this model and the use of relevant influenza viruses, known to be or likely to be of high virulence in humans, provides a unique opportunity to elucidate this important aspect of avian influenza infection in humans. Bielefeldt Ohmann - R03 NARRATIVE Outbreaks of avian influenza in humans have caused increasingly high morbidity and mortality. Further adaptation of the virus to the human host is likely to result in a world-wide epidemic such as the 1918 or "Spanish" influenza, believed to have been of avian origin and to have caused 20 to 40 millions deaths worldwide. Human disease caused by infection with avian influenza is not limited to the respiratory tract, but appears to affect other organs in ways that result in poor prognosis in infected individuals. We propose to perform in-depth pathology work in non-human primates that are to be infected with a highly pathogenic avian virus and with a human virus recombined with genes from the 1918 influenza, in order to fully characterize the effect of infection on tissues outside of the respiratory tract and with the goal of contributing to the body of knowledge that will help minimize human casualties after infection. [unreadable] [unreadable] [unreadable]